Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan, United States of America.
PLoS Comput Biol. 2013;9(11):e1003279. doi: 10.1371/journal.pcbi.1003279. Epub 2013 Nov 21.
DnaK is a molecular chaperone that has important roles in protein folding. The hydrolysis of ATP is essential to this activity, and the effects of nucleotides on the structure and function of DnaK have been extensively studied. However, the key residues that govern the conformational motions that define the apo, ATP-bound, and ADP-bound states are not entirely clear. Here, we used molecular dynamics simulations, mutagenesis, and enzymatic assays to explore the molecular basis of this process. Simulations of DnaK's nucleotide-binding domain (NBD) in the apo, ATP-bound, and ADP/Pi-bound states suggested that each state has a distinct conformation, consistent with available biochemical and structural information. The simulations further suggested that large shearing motions between subdomains I-A and II-A dominated the conversion between these conformations. We found that several evolutionally conserved residues, especially G228 and G229, appeared to function as a hinge for these motions, because they predominantly populated two distinct states depending on whether ATP or ADP/Pi was bound. Consistent with the importance of these "hinge" residues, alanine point mutations caused DnaK to have reduced chaperone activities in vitro and in vivo. Together, these results clarify how sub-domain motions communicate allostery in DnaK.
DnaK 是一种分子伴侣，在蛋白质折叠中具有重要作用。ATP 的水解对这种活性至关重要，核苷酸对 DnaK 结构和功能的影响已经得到了广泛的研究。然而，控制构象运动的关键残基，从而定义 apo、ATP 结合和 ADP 结合状态，尚不完全清楚。在这里，我们使用分子动力学模拟、突变和酶测定来探索这个过程的分子基础。在 apo、ATP 结合和 ADP/Pi 结合状态下，对 DnaK 的核苷酸结合结构域 (NBD) 的模拟表明，每种状态都具有独特的构象，与可用的生化和结构信息一致。模拟进一步表明，亚结构域 I-A 和 II-A 之间的大剪切运动主导了这些构象之间的转换。我们发现，几个进化上保守的残基，特别是 G228 和 G229，似乎作为这些运动的铰链，因为它们主要根据是否结合 ATP 或 ADP/Pi 而存在于两个不同的状态。这些“铰链”残基的重要性与实验结果一致，即 DnaK 的丙氨酸点突变导致其在体外和体内的伴侣活性降低。总之，这些结果阐明了亚结构域运动如何在 DnaK 中传递变构。
